The AMPA type ionotropic glutamate receptors (AMPARs), a ligand gated ion channel activated by the neuro- transmitter glutamate, mediate the majority of excitatory neurotransmission in the brain. The signals trans- duced by these complexes are critical for synaptic plasticity, learning and memory. AMPAR auxiliary subunits regulate trafficking and gating modulation of AMPARs. In this proposal we will investigate the mechanism of AMPAR regulation by their auxiliary subunits. The two major AMPAR auxiliary subunits, in the hippocampus, cortex, and striatum, are TARPs and cornichons (CNIHs). The TARPs are extensively studied and therapeutic compounds to alleviate seizure are already available to target ?-8 TARP, a hippocampus enriched TARP. On the other hand, our understanding on CNIHs is limited. Within the CNIH family, CNIH2/3 is known to function as AMPAR auxiliary subunits. In humans, the N-terminus of CNIH2 that forms the interaction interface with AMPAR is intolerant to missense mutations, indicating an essential role of CNIH2-AMPAR interaction in hu- mans. Our hypothesis is that CNIHs play fundamental roles in regulating AMPAR gating during synaptic transmission and plasticity. To further establish this hypothesis, we will study the functional mechanism of complexes made of GluA2 subunit of AMPAR and CNIH3 as a model. Our lab has recently solved the cryo-EM structure of GluA2/CNIH3 complex in GluA2:CNIH3=4:4 stoichiometry at high resolution. In Aim 1 we hypothe- size that the GluA2/CNIH3 complex could exists in other stoichiometry, and propose to reveal the architecture of complex in GluA2:CNIH3=4:2 stoichiometry using cryo-EM. CNIH1 is currently not categorized as AMPAR auxiliary subunit. However the cryo-EM structure of the GluA2/CNIH3 complex tells us that CNIH1 possess AMPAR binding motif that is present in CNIH2/3. The cryo-EM structure also revealed the presence of lipids surrounding the complex. We hypothesize that these lipids may play important functional roles in AMPAR gat- ing modulation. In Aim2 we will test roles of CNIH1 and lipids in gating modulation of AMPAR. Finally, we hy- pothesize that revealing the allosteric gating modulation mechanism of CNIH3 would require obtaining snap- shots of lipid embedded GluA2/CNIH3 complex in channel closed, open, and desensitized states. In Aim 3, we propose to solve high resolution cryo-EM structures of GluA2/CNIH3 complex embedded in a lipid bilayer mi- metic environment, and compare them in different functional states. The role of auxiliary subunits in tuning ion channel gating kinetics is predicted to have significant impact on circuit dynamics. In summary, the outcomes of this study are expected to advance our mechanistic understanding of AMPAR function and assist developing new therapeutic compounds that can alleviate dysregulation of AMPARs seen in neurological and psychiatric disorders, such as Alzheimer's disease, stroke, autism, Rasmussen's and limbic encephalitis, and seizure.